Quorum sensing orchestrates parallel cell death pathways in Vibrio cholerae via Type 6 secretion dependent and independent mechanisms

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In Vibrio cholerae colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type VI secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control t6ss gene expression nor T6SS-mediated killing activity. Nonetheless, a {Delta}hapR strain displays no cell death at the colony rim. RNA-Seq analyses reveal that HapR activates expression of an operon containing four genes of unknown function, vca0646-0649. Epistasis and overexpression studies show that two of the genes, vca0646 and vca0647, are required to drive cell death in both a {Delta}hapR and a {Delta}hapR {Delta}t6ss strain. Thus, vca0646-0649 are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in V. cholerae. SignificanceCell death is a fundamental biological process. In mammals, cell death sculpts tissues during development, enables injury recovery, and regulates immunity. In bacteria, cell death mechanisms remain little explored. Recently, colonies formed by the pathogen Vibrio cholerae were demonstrated to undergo a spatio-temporal program of cell death. The program is controlled by quorum sensing (QS) and driven by the Type VI secretion system. Here, we discover QS-controlled genes, called vca0646-0649, that cause cell death in V. cholerae colonies independently of the Type VI secretion system. These findings indicate that a second cell death pathway exists in V. cholerae. The results expand our understanding of bacterial cell death mechanisms and provide insight into how cell death shapes bacterial community structure.